We are utilizing bioinformatics/evolutionary conservation analysis to determine if microRNAs (miRNAs) may target the interferon-gamma mRNA. To address this possibility, we have generated a stable small interfering RNA (siRNA) transfectant of the dicer gene in the human natural killer cell line NK92. Dicer is an enzyme critical for overall miRNA processing and thus decreased dicer levels may alter interferon-gamma gene expression if miRNAs are involved in gene expression. Results indicate that interferon-gamma expression may be altered in response to specific stimuli in the dicer knockdown NK92 transfectant, thus implicating miRNAs in the regulation of interferon-gamma expression. Furthermore, we have identified an evolutionarily conserved miRNA binding site in the interferon-gamma 3'untranslated region and preliminary data indicates that this site contributes to regulating interferon-gamma expression. Binding of this miRNA to the IFN-gamma mRNA may result in an altered conformation of the RNA, resulting in increased IFN-gamma expression due to a stabilization of the mRNA. Such a stabilization would represent a new model for the role of a miRNA in regulating IFN-gamma gene expression. In addition to the IFN-gamma gene, we are investigating the possible role of miRNAs in regulating HLSA-C gene expression, in collboration with the laboratory of Dr. Mary Carrington. Preliminary results have identified a miRNA binding site in the 3'untranslated region of the gene and polymorphisms have been identified in this region. current efforts are focused on characterizing the role of this miRNA binding site in HLA-C cell surface expression and the impact of the SNPs on this gene expression. As part of this overall project, we are also searching for polymorphisms in the IFN-gamma gene that may effect gene expression by altering IFN-g mRNA stability.